In mammals, the X-chromosome is uniquely capable of global transcriptional inactivation. X-inactivation enables females (XX) and males (XY) to achieve equal-X chromosome dosage despite unequal X-chromosome number (dosage compensation). Since X-inactivation is a large-scale silencing phenomenon, an understanding of its mechanism would establish new paradigms for long-range chromatin effects. Because X-inactivation plays an important role during development and is believed to occur during spermatogenesis, its mechanism would also have significant implications for developmental biology and medical science. At present, only two elements are known to be involved in control of X-inactivation. The "X- inactivation center" (Xic) is located on the X-chromosome and as a master switch to turn on the silencing process. A required gene called Xist resides at the Xic and encodes an untranslated RNA which acts in cis to coat the inactive X. Previous transgenic work suggested that the Xic may be entirely contained within a 450 kb sequence carried on a yeast artificial chromosome (YAC). However, the molecular mechanisms of Xic/Xist action remain unknown. Nor is it known what other elements participate in the process. A functional dissection of the switch region and identification of additional regulatory factors form the objectives of this proposal. The research plan is based on a YAC transgenic assay which allows for genetic analysis in mouse embryonic stem cells. Two types of analyses will be pursued. First, large-scale genomic deletions will be constructed across the 450 kB YAC and analysed in transgenic mouse cells to identify critical subregions for X-inactivation. Second, a panel of overlapping Xist gene deletions will be created on the YAC to identify important domains for Xist function. Once required Xic subregions and Xist domains are uncovered, effort will be directed towards cloning of essential elements and isolation of interacting factors.